This invention relates to a method of amplifying production of IgG, IgA and IgM antigen-specific human monoclonal antibodies from Epstein Barr virus (EBV) transformed, T-cell depleted human peripheral blood lymphocytes through the use of an adjuvant system consisting of 8-mercaptoguanosine and at least one of the cytokines interleukin-4 (IL-4) and interleukin-6 (IL-6). It also includes the adjuvant system and a kit comprising such system.
Monoclonal antibodies derived from mice are the reagents most commonly used in in vivo therapeutic and diagnostic procedures and for in vitro diagnostic testing. Although a good deal of success has been had with the use of these murine monoclonals, a major disadvantage is that they are not identical to antibodies produced by humans. Because of the species differentiation, when use is made of murine monoclonal antibodies in the in vivo diagnostic or therapeutic treatment of humans, it is now known that anti-murine antibodies may be produced in the treated patient. The presence of these anti-murine antibodies can sensitize the patient to the degree that additional therapy or in vivo diagnostic testing using murine monoclonal antibodies is precluded. In vitro test results on sensitized patients also may be rendered erroneous due to interactions of the anti-murine antibodies with the murine antibodies in the diagnostic assay. In order to eliminate these problems, the monoclonal antibody of choice, especially for use in vivo, is one derived from humans.
Although it is theoretically possible to produce human antibodies using cells from various organs, such as the spleen or the tonsils, the most readily available souce is the peripheral blood supply.
It is known to those skilled in the art that it is possible to produce antigen specific monoclonal antibodies derived from human peripheral blood lymphocytes (PBL). Unfortunately, the success rate for producing antigen specific human antibodies of the right isotype is difficult and particularly time consuming. The majority of research on monoclonal antibody production has been performed on the murine model, and the use of PBL is a newer field. Since the donor generally is not and cannot be immunized prior to donating blood, the B cells from the PBL must be exposed to the proper antigen, and thereby activated, only in vitro. The activation by the antigen begins the cycle that continues with proliferation of the B cells and then differentiation of these cells into antibody producing cells. Once these cells produce antibodies to the antigen, it has generally been found that the IgM isotype is the isotype most commonly produced. However, the isotype of choice for many applications is IgG.
It is well known that presentation of antigen in vitro is very difficult to do technically. Enhancement of antigen specific B cell activation, proliferation and differentiation is known to occur with a class of compounds known as C8-substituted guanine ribonucleosides (M. G. Goodman, "Immunobiologic properties of the C8-Derivatized Guanine Ribonucleosides", Biomedicine & Pharmacotherapy, 37, 344-350 (1983); M. G. Goodman et al., "Derivatized Guanine Nucleosides: A New Class of Adjuvant for In Vitro Antibody Responses", J. of Immuno., 130, 2580-2585 (1983); L. Danielsson et al., "Effect of Cytokines on Specific In Vitro Immunization of Human Peripheral B Lymphocytes Against T-cell Dependent Antigens", Immunology, 61, 51-55 (1987); M. G. Goodman, "Role of Salvage and Phosphorylation in the Immunostimulatory Activity of C8-Substituted Guanine Ribonucleosides", J. Immuno., 141, 2394-2399 (1988); W. J. Hennen et al., EPA 0 341 065, "Immunostimulating Guanine Derivatives, Compositions and Methods".) Some of the more active compounds are 8-bromoguanosine, 8-mercaptoguanosine and 7-methyl-8-oxoguanosine. Although the specific mode of activation is not precisely known, the C8-derivatized guanine ribonucleosides, and in particular, 8-mercaptoguanosine (8-MG), have the capability to enhance B-cell activation to produce an antigen specific antibody producing cell. Thus, while the number of antigen specific B-cells present in PBL at the initiation of culture may be less than 1 in 10.sup.6 -10.sup.7 cells, the addition of 8-MG allows preferential clonal expansion of those cells responding to antigen in vitro.
L. Danielsson et al. (see above) have investigated the effect of cytokines in the antigen specific activation of human B lymphocytes. Both T-cell depleted and unseparated PBL were used in conjunction with haemocyanin as the antigen and mouse recombinant IL-1 and human recombinant IL-2, along with other cytokines as the adjuvants, in this study. It was discovered that no in vitro immunization occurred in unseparated PBL. In T-cell depleted PBL, antigen specific human B cells were found and differentiation was improved with the addition of B cell differentiation factor and IL-2. Addition of IL-1 had only a negligible effect. However, the use of a B cell differentiation factor, in this case, pokeweed mitogen T-cell replacing factor, was absolutely necessary to obtain any response at all.
In 1985, Goodman and Weigle studied the antigen specific primary antibody response of T-cell depleted PBL in vitro, using 7-methyl-8-oxoguanosine and IL-2. The results show that this combination of adjuvants in a T-cell depleted PBL system generated an antigen specific antibody response. 7-methyl-8-oxoguanosine was tested as the only adjuvant, as was 8-MG. While 7-methyl-8-oxoguanosine consistently induced a high degree of enhancement of the antibody response, 8-MG induced a much smaller response. (M. G. Goodman et al., "Enhancement of the Human Antibody Response by C8-Substituted Guanine Ribonucleosides in Synergy with Interleukin 2", J. Immuno., 135, 3284-3288 (1985)).
Later, a similar study was done in order to determine if various cytokines were able to synergise with 8-MG to augment B cell responsiveness to antigen. Murine cell lines were used. The data indicated that synergistic effects did not occur with interferon-gamma, purified IL-1, IL-2, IL-3, IL-4 or IL-5, but did with interferon-alpha and interferon-beta. (M. G. Goodman, "Interaction Between Cytokines and 8-Mercaptoguanosine in Humoral Immunity: Synergy with Interferon", J. Immuno., 139,142-146 (1987)).
Another problem facing investigators attempting to raise human monoclonals from PBL is the need for IgG and IgA isotypes. Efforts to immortalize antigen specific antibody producing B cells use traditional protocols, such as transformation of these cells with EBV. Unfortunately, EBV transformed cells produce antibodies preferentially of the IgM isotype. A method for producing human polyclonal antibodies containing substantially less IgM and substantially more IgG and IgA than is currently known is needed. These human monoclonal antibody producing cells could then be harvested, cloned and used to produce human monoclonal antibodies in cell culture.